Summary/Abstract Many diseases of the brain involve cellular stress. Early in life, the Zika virus triggers stress in neural progenitor cells causing microcephaly. Much later in life the mutant proteins produced in Parkinson's and Huntington's disease, as well as ALS, and Alzheimer's all produce cellular stress leading to the slow neurodegeneration that occurs over many years. Typically this degeneration is studied by counting dead cells, in animal or cellular models of the disease, which is an insensitive measurement of chronic, slow, cellular stress. Our goal is to create a genetically encoded, fluorescent biosensor that lights up living, stressed cells long before they are destined to die, giving scientists sensitive new tools to identify and potentially rescue stressed neurons. Aim one will test a series of multicolored sensor prototypes designed to measure cellular stress as well as second messenger signaling. These sensors will be tested in standard cell lines. Comparison of the sensor responses in cells expressing normal or disease forms of neurodegenerative proteins will be used to identify the best possible sensor designs. The broad suite of mutant proteins being examined will give us a first glimpse of whether one specific sensor is most sensitive, or whether different sensors are best suited to study different stressors and diseases. Aim two will test whether these tools can detect stress in neurons. The first goal will be to detect stress caused by overexpression of the normal and mutant proteins from aim one in neuronal culture as well as in neurons in rat brain slices. The second goal will be to use iPSC derived motor neurons from a patient with ALS, as well as an isogenic cell line in which the SOD1 mutation from the patient has been repaired. If the sensor can detect the difference, this will be a significant improvement over existing technologies.